Wnt signaling determines cell fates during embryonic development in all animals, including humans, and is required later in life to maintain stem cell populations. Excess Wnt activity is associated with human cancers, particularly colon cancer. To understand how Wnt signaling causes cancer, the factors that normally control Wnt pathway activity must be fully characterized. The long term objective of the Bejsovec laboratory is to dissect the mechanics of this important signaling pathway, using both the Drosophila genetic model system and cultured human cells. This lab has identified by mutational analysis many important pathway components, such as Tcf, the transcription factor that activates Wnt target genes, and Apc2, a human tumor suppressor homolog that negatively regulates the Wnt pathway. This proposal focuses on another negative regulatory gene identified in the same mutational screen: tumbleweed (tum) is a homolog of the human RacGap1, a GTPase activating protein (GAP). Surprisingly, the GAP activity of this gene is essential for cell division, but its GAP activity is not required for its role in Wnt regulation. Two Tum-interacting molecules, identified in yeast two- hybrid screens, strongly modulate Wnt pathway activity in human cells. Pavarotti (Pav), a kinesin-like protein, acts as a repressor in a similar fashion to Tum, while the other interactor, a novel fly protein tentatively named Sills, enhances Wnt-induced target gene expression. This application proposes three aims to determine the mechanism of action for Tum and its binding partners. Both Tum and Pav play a Wnt-independent role in organizing the cytoskeleton during cell division, so a primary issue is whether their Wnt- dependent role also occurs in the cytoplasm. Aim 1 will determine the subcellular locations relevant to their Wnt-modulating activity. Aim 2 will address whether Tum and Pav interact directly with other Wnt pathway components or with the transcription complex that forms on Wnt target gene promoters. Aim 3 will characterize the cellular function of Sills and its human homolog, using both fly embryos and cultured cells to determine how it enhances Wnt activity. These experiments will help formulate a clearer picture of how living tissue modulates the response to Wnt signal. PUBLIC HEALTH RELEVANCE: Wnt signaling is essential for normal development in all animals, but abnormal Wnt signaling is associated with various forms of cancer, particularly colorectal cancer. Experiments proposed here will use both the fruitfly model system and cultured human cells to study newly-discovered genetic factors that regulate Wnt signaling.